Antigen-specific helper t-cell receptor genes

ABSTRACT

The present invention pertains to polynucleotides that encode CDR3 in TCR-[alpha] and TCR-[beta] chain genes of CD4 +  helper T-cells that are specific to WT1 helper peptides having an amino acid sequence represented by SEQ ID NO: 123. The present invention further pertains to the peptides encoded by said polynucleotides. The present invention further pertains to CD4 +  T cells into which TCR genes that contain said polynucleotides have been introduced, the induction of WT1-specific cytotoxic T-lymphocytes (CTLs) using the CD4 +  T-cells, the treatment of cancer, etc.

TECHNICAL FIELD

The present invention relates to polynucleotides contained in T cellreceptor (TCR) genes of cancer antigen-specific helper T-cells. Inparticular, the present invention relates to polynucleotides encodingcomplementarity determining region 3 (CDR3) of each α-chain and β-chainof TCR of CD4⁺ helper T-cells specific to a WT1 helper peptide having anamino acid sequence shown in SEQ ID NO: 123. The present invention alsorelates to polypeptides encoded by these polynucleotides. Further, thepresent invention relates to CD4⁺ T-cells into which TCR genescontaining these polynucleotides are introduced, induction andenhancement of WT1-specific cytotoxic T cells (WT1-specific CTL) usingthem, and treatment of cancers using them, and so on.

BACKGROUND ART

WT1 gene (Wilms' tumor 1 gene) is a gene which was identified as a generesponsible for Wilms tumor which is a renal cancer in children(Non-patent Documents 1 and 2). WT1 is a transcription factor having azinc finger structure. Initially, WT1 gene was considered to be a tumorsuppressor gene, However, subsequent studies (Non-patent Documents 3, 4,5 and 6) showed that WT1 gene rather functions as an oncogene inhematopoietic tumors and solid cancers.

It was showed that peptide-specific cytotoxic T cells (CTLs) wereinduced by stimulating peripheral mononuclear cells in vitro using a WT1peptide and that these CTLs damage tumor and cancer cells inhematopoietic tumors and solid cancers which express WT1 endogenously.Because CTL recognizes a WT1 peptide in a form of a complex in which theWT1 peptide is bound to a MHC class I molecule, such a WT1 peptidediffers in accordance to MHC class I subtype (Patent Document 1,Non-Patent Document 7, Patent Documents 2, 3 and 4).

Existence of helper T cells specific to a cancer antigen is importantfor effective induction of CTL (Non-Patent Document 8). Helper cells areinduced and activated by recognizing a complex of a MHC class IImolecule and an antigen peptide on antigen-presentino cells. Activatedhelper T cells produce cytokines such as IL-2, IL-4, IL-5, IL-6, orinterferons, and help proliferation, differentiation and maturation of Bcells. Activated helper T cells also have a function to promoteproliferation, differentiation and maturation of other subset of T cells(such as Tc cells). Thus, because activated helper T cells have afunction to activate immune system by promoting proliferation andactivation of B cells and T cells, it is considered that enhancing thefunction of helper T cells via a MHC class II binding antigen peptide(helper peptide) to enhance the effect of a cancer vaccine useful incancer immunotherapy (Non-Patent Document 9).

Examples of helper peptides relating to WT1 which are presentlyrecognized are a peptide binding to HLA-DRB1*04:01 molecule (Non-PatentDocument 10), a peptide binding to HLA-DEB1*04:05 molecule, a peptidebinding to HLA-DRE1*15:02 molecule (Patent Document 5), a peptidebinding to HLA-DRB1*04:05 molecule, HIA-DRB1*15:02 molecule,HLA-DRB1*15:01 molecule, HILA-DPB1*09:01 molecule and HLA-DPB1*05:01molecule (Patent Document 6).

However, sequences of T cell receptor (TCR) genes of antigen-specificCD4⁺ helper T-cells which recognize a helper peptide have not been knownat all.

DOCUMENTS OF BACKGROUND ART Patent Documents

Patent Document 1: WO2003/106682

Patent Document 2: WO2005/095598

Patent Document 3: WO2007/097358

Patent Document 4: PCT/JP2007/074146

Patent Document 5: WO2005/045027

Patent Document 6: WO2008/105462

Non-Patent Documents

Non-Patent Document 1: Daniel A. Haber et al., Cell. 1990 Jun. 29;61(7):1257-69.

Non-Patent Document 2: Call K M et al., Cell. 1990 Feb. 9; 60(3):509-20.

Non-Patent Document 3: Menke A L et al., Int Rev Cytol. 1998;181:151-212. Review.

Non-Patent Document 4: Yamaqami et al., Blood. 1996 Apr. 1;87(7):2878-84.

Non-Patent Document 5: Inoue K et al., Blood. 1998 Apr. 15;91(8):2969-76.

Non-Patent Document 6: Tsuboi A et al., Leuk Res. 1999 May;23(5)499-505.

Non-Patent Document 7: Oka Y et al,, Immunogenetics. 2000 February;51(2):99-107.

Non-Patent Document 8: Gao F G et al,, Cancer Res. 2002 Nov. 15;62(22):6438-41.

Non-Patent Document 9: Zeng G, J Immunother. 2001 May; 24(3):195-204.

Non-Patent Document 10: Knights A J et al., Cancer Immunol Immunother2002 July; 51(5)271-81.

SUMMARY OF INVENTION Problem to be Solved by the Invention

Problems to be solved by the invention were to determine sequences ofTCR genes of CD4⁺ helper T-cells specific to a WT1 helper peptide, toobtain CD4⁺ T-cells into which these ICR genes have been introduced, toenhance induction of WT1 specific CTL using such cells, and to treat orprevent cancers, and so on.

Means to Solve the Problem

The inventors studied vary hard to solve the above problems, andsucceeded in isolating α-chain genes and β-chain genes of TCR of CD4⁺helper T-cells specific to a WT1 helper peptide, and determined eachCDR3 sequence. Further, the inventors introduced TCR genes containingthe sequences thus determined into CD4⁺ T-cells, and succeeded inenhancing induction of WT1 specific CTL and damaging WT1 expressingcancer cells by use of the CD4⁺ T-cells. Thus, the inventors havecompleted the present invention.

That is, the present invention provides:

-   (1) A polynucleotide (referred as “αCDR3 polynucleotide”) having a    nucleotide seauence selected from the group consisting of SEQ ID    NOs: 1, 3, 5, 8, 10, 11, 13, 14, 16, 18, 20, 22, 23, 25, 27, 20, 30,    31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 56 and 58,    wherein said polynucleotide encodes CDR3 of α-chain of TCR of a CD4⁺    helper T-cell specific to a WT1 helper peptide (WT1₃₃₂ peptide)    havind an amino acid sequence shown in SEQ ID NO: 123 or a variant    sequence thereof.-   (2) A polynucleotide (referred as “βCDR3 polynuclectide”) a    nucleotide sequence selected from the group consisting of SEQ ID    NOs: 2, 4, 6, 7, 9, 12, 15, 17, 19, 21, 24, 26, 29, 32, 34, 36, 38,    40, 42, 44, 46, 48, 50, 52, 54, 57 and 59, wherein said    polynucleotide encodes CDR3 of β-chain of TCR of a CD4⁺ helper    T-cell specific to WT1₃₃₂ peptide.-   (3) A pair of a αCDR3 polynucleotide and a βCDR3 polynucleotide,    wherein each polynucleotide has the following nucleotide sequence:

α CDR3 polynucleotide βCDR3 polynucleotide SEQ ID NO: 1 SEQ ID NO: 2 SEQID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 3 SEQ ID NO:7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 12 SEQ ID NO: 11SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 15 SEQ ID NO: 14 SEQ ID NO: 15SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 24 SEQ ID NO: 23 SEQ ID NO: 24SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 4 SEQ ID NO: 28 SEQID NO: 29 SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 31 SEQ ID NO: 32 SEQ IDNO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ IDNO: 38 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ IDNO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ IDNO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ IDNO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 57 SEQ ID NO: 56 SEQ IDNO: 57 SEQ ID NO: 58 SEQ ID NO: 59provided that said sequence may be a complementary sequence or adegenerate sequence thereof.

-   (4) A TCR gene comprising the αCDR3 polynucleotide and the βCDR3    polynucleotide in any one of pairs of (3).-   (5) The TCR gene of (4) obtainable from a CD4⁺ T-cell specific to    WT1₃₃₂ peptide.-   (6) A method for producing a CD4⁺ helper cell specific to WT1₃₃₂    peptide, comprising introducing the TCR gene of (4) into a CD4⁺    cell.-   (7) A CD4⁺ helper T-cell obtainable by the method of (5).-   (8) A vector comprising a TCR gene which comprises the αCDR3    polynucleotide and the βCDR3 polynucieotide in any one of pairs of    (3).-   (9) The method of (6) wherein said introduction is performed using    the vector of (8).-   (10) A method for enhancing the induction of a WT1 specific CTL,    comprising co-culturing the CD4⁺ helper T-cell of (7) and a    peripheral mononuclear cell.-   (11) A WT1-specific CTL obtainable by the method of (10).-   (12) A method for the treatment or prevention of a cancer in a    subject, comprising introducing the CD4⁺ helper T-cell of (7) into    the subject.-   (13) A pharmaceutical composition for the treatment or prevention of    a cancer, comprising the CD4⁺ helper T-cell of (7).-   (14) Use of the CD4⁺ helper T-cell of (7) for the manufacture of a    medicine for the treatment or prevention of a cancer.-   (15) A DNA chip comprising the nCDR3 polynucleotide of (1), the    βCDR3 polynucleotide of (2), or both of the αCDR3 polynucleotide    of (1) and the βCDR3 polynucleotide of (2).-   (16) A method for measuring the frequency of CD4⁺ helper T-cell    specific to WT1₃₃₂ peptide in a sample, comprising using the DNA    chip of (15).-   (17) An αCDR3 peptide encoded by any one of the αCDR3    polynucleotides of (1). (18) A βCDR3 peptide encoded by any one of    the βCDR3 polynucleotides of (2).-   (19) A pair of peptides encoded by any one of pairs of the    polynucleotides of (3).-   (20) A chip comprising the peptide of (17) or (18), or the pair of    the peptides of (19).-   (21) An antibody against any one of the peptides of any one of    (17)-(19).-   (22) A method for measuring the frequency of CD4⁺ helper T-cell    specific to WT1₃₃₂ peptide in a sample, comprising using the    antibody of (21).

EFFECT OF THE INVENTION

According to the present invention, a CD4⁺ helper T-cell is obtainedinto which a TCR gene having the CDR3 nucleotide sequence determined bythe present invention has been introduced. A. WT1-specific CTL can beinduced using said CD4⁺ helper T-cell, and a cancer can be treated orprevented effectively, Further, a DNA chip is prepared using the TCRsequences, and frequency of WT1₃₃₂-specific CD4⁺ helper T-cells in asample can be measured.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows nucleotide sequences of CDR3 α-chains and β-chains of TCRof CD4⁺ helper T-cells obtained by the present invention, and amino acidsequences of CDR3 encoded thereby. Number in parentheses positioned atthe right end of each sequence presents SEQ ID NOs in SEQUENCE LISTING.V-GENE, J-GENE and J-GENE describe V region, J region and D region inindividual genes, respectively.

FIG. 1B shows nucleotide sequences of CDR3 α-chains and β-chains of TCRof CD4⁺ helper T-cells obtained by the present invention, and amino acidsequences of CDR3 encoded thereby. Number in parentheses positioned atthe right end of each sequence presents SEQ ID NOs in SEQUENCE LISTING,V-GENE, J-GENE and J-GENE describe V region, J region and region inindividual genes, respectively.

FIG. 2A shows interferon-γ production by WT1₃₃₂-specific CD4⁺ helperT-cells into which TCR genes shown in Table 3 have been introduced.

FIG. 2B shows IL-2 production , WT1₃₃₂-specific CD4⁺ helper T-cells intowhich TCR genes shown in Table 3 have been introduced.

FIG. 2C shows TNF-α production response of WT1₃₃₂-specific CD4⁺ helperT-cells into which TCR genes have been introduced, to WT1₃₃₂ peptideconcentration.

FIG. 2D shows proliferation ability of WT1₃₃₂-specific CD4⁺ helperT-cells into which TCR genes have been introduced, in systems into whichseveral kinds of substances are added. WT1₃₃₂ represents the culture inthe presence of WT1₃₃₂ peptide α-DP represents the culture in thepresence of an anti-HLA-DP antibody. α-DQ represents the culture in thepresence of an anti-HLA-DQ antibody, α-DR represents the culture in thepresence of an anti-HLA-DR antibody. HLA class I represents the culturein the presence of an anti-HLA class I antibody. HIV represents theculture in the presence of HIV peptide (FRKQNPDIVIYQYMDDLYVG)(SEQ ID NO:124).

FIG. 2E shows proliferation of WT1₃₃₂-specific CD4⁺ helper T-cells intowhich TCR genes have been introduced, in the presence of PBMC pulsedwith several kinds of substances. WT1₃₃₂ represents the stimulation byautologous PBMC pulsed with WT1₃₃₂ peptide. HWT1 represents thestimulation by autologous PBMC pulsed with full length of WT1 protein.HWT3 represents the stimulation by autologous PBMC pulsed with truncatedWT1 protein (not containing WT1₃₃₂ sequence). PHA-blast represents thestimulation by PBMC pulsed with PHA-blast lysate. TF1 represents thestimulation by PBMC pulsed with a lysate of leukemic cell line TF-1expressing WT1. K562 represents the stimulation by PBMC pulsed with alysate of leukemic cell line K562 expressing WT1.

FIG. 2F shows IFN-γ production by WT1₃₃₂-specific CD4⁺ helper T-cellsinto which TCR genes have been introduced, in the presence of PBMCpulsed with several kinds of substances, WT1₃₃₂ represents thestimulation by autologous PBMC pulsed with WT1₃₃₂ peptide. HWT1represents the stimulation by autologous PBMC pulsed with full length ofWT1 protein. HWT3 represents the stimulation by autologous PBMC pulsedwith truncated WT1 protein (not containing WT1₃₃₂ sequence). PHA-blastrepresents the stimulation by PBMC pulsed with PHA-blast lysate, TF1represents the stimulation by PBMC pulsed with a lysate of leukemic cellline TF-1 expressing WT1. K562 represents the stimulation by PBMC pulsedwith a lysate of leukemic nein line K562 expressing WT1.

FIG. 2G shows the average of the production of several kinds o cytokinesin response to Wr1₃₃₂ peptide by WT1₃₃₂-specific CD4⁺ helper- T-celllines from three healthy subjects (HLA-DPB1*05:01-positive) into whichTCR genes have been introduced. Black bars represent with thestimulation by WT1₃₃₂ peptide. White bars represent without thestimulation.

FIG. 3A shows the frequency of CD3⁺CD8⁺T cells in case PBMO andWT1₃₃₂-specific CD4⁺ helper T-cells into which TCR genes have beenintroduced are co-cultured at the ratio as shown in the figure.

FIG. 3B shows the frequency of the modified WT1₂₃₅/HLA-A*24:02tetramer-positive CD8⁺T cells in case PBMC and WT1 ₃₃₂-specific CD4⁺helper T-cells into which TCR genes have been introduced are co-culturedat the ratio as shown in the figure.

FIG. 3C shows the cell number of WT1 specific. CTL in case PBMO andWT1₃₃₂-specific CD4⁺ helper T-cells into which TCR genes have beenintroduced are co-cultured at the ratio as shown in the figure.

FIG. 3D shows the frequency of CD8⁺T cells expressing interferon-γ inresponse to the stimulation by the modified WT1₂₃₅ in case PBMC andWT1₃₃₂-specific CD4⁺ helper T-cells into which TCR genes have beenintroduced are co-cultured at the ratio as shown in the figure.

FIG. 4A shows the damage as the lysis (%) of HLA-DFB1*05:01-positiveleukemic cell line TF-1 expressing WT1 and HLA-DRB1*05:01-negativeleukemic cell line TF-1 expressing WT1 by WT1₃₃₂ specific CD4⁺ helperT-cells into which TCR genes have been introduced at the E:T ratio asshown in the figure.

FIG. 4B shows the damage as the lysis (%) of HLA-DPB1*05:01-positiveB-LCL cells which have been enforced to express WT1 andHLA-DPB1*05:01-positive B-LCL cells which do not express WT1 byWT1₃₃₂-specific CD4⁺ helper T-cells into which TCR genes have beenintroduced at the E:T ratio as shown in the figure.

FIG. 4C shows the damage as lysis (%) of K562 cell line andHLA-DPB1*05:01-positive leukemic cell line C2F8 expressing WT1 byWT1₃₃₂-specific CDC helper T-cells into which TCR genes have beenintroduced at the E:T ratio as shown in the figure.

FIG. 4D shows the results by flow-cytometry for the expression ofGranzymeB (left) and Perfolin (right) in WT1₃₃₂-specific CD4⁺ helperT-cells into which TCR genes have been introduced.

FIG. 4E shows the results by flow-cytometry for the frequency of CD107aproducing cells and IFN-γ producing cells of WT1₃₃₂-specific CD4⁺ helperT-cells into which TCR acnes have been introduced, cultured according tothe method described in Example 4.

FIG. 4F is a bar graph showing the results for the comparison of thecell damaging activity of WT1₃₃₂-specific CD4⁺ helper T-cells into whichTCR genes have been introduced against HLA-CPB1*05:01-positive TF-1cells pretreated with Ac-IETD-Cho, with the cell damaging activity ofWT1₃₃₂-specific CD4⁺ helper T-cells into which TCR aenes have beenintroduced against TF-1 cells pretreated with DMSO. Height of barsrepresents the average value with bars of standard deviation. Asteriskrepresents p<0.05.

FIG. 5 shows survival curves showing anti-tumor activity in NOG(Registered Trade Mark) mice by human CD4⁺ T-cells into which TCR genesfrom WT1₃₃₂ specific CD4⁺ T-cells have been introduced. The solid linerepresents the survival curve of mice into which human CD4⁺ T-cells havebeen transferred, into which HLA-DPB1*05:01 restricted WT1₃₃₂-specificTCR have been introduced. The broken line represents the survival curveof mice into which human CD4⁺ T-cells have been transferred, into whicha control vector have been introduced.

DESCRIPTION OF EMBODIMENTS

The present invention is based on the determination of thepolynucleotides encoding α-chain containing CDR3 (hereinafter referredas “αCDR3 polynucleotide”) and the polynucleotides encoding β-chaincontaining CDR3 (hereinafter referred as “βCDR3 polynucleotide”) of TCRof CD4⁺ helper T-cell clones specific to a WT1 helper peptide. Thus, inone aspect, the present invention provides αCDR3 polynucleotides havingnucleotide sequences shown in FIG. 1 (nucleotide sequences selected fromthe group consisting of SEQ ID NOs: 1, 3, 5, 8, 10, 11, 13, 14, 16, 18,20, 22, 23, 25, 27, 28, 30, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 56, 58), and βCDR3 polynucleotides having nucleotide sequencesshown in FIG. 1 (nucleotide sequences selected from the group consistingof SEQ ID NOs: 2, 4, 6, 7, 9, 12, 15, 17, 19, 21, 24, 26, 29, 32, 34,36, 38, 40, 42, 44, 46. 48, 50, 52, 54, 57, 59).

Preferably, from the viewpoint of expression of receptor function, anαCDR3 polynucleotide and a βCDR3 polynucleotide contained in each cloneare contained in one TCR. That is, it is preferable that an αCDR3polynucleotide and a βCDR3 polynucleotide corresponding to each cloneform a pair as shown in FIG. 1. Therefore, in a further aspect, thepresent invention provides a pair of a αCDR3 polynucleotide and a βCDR3polynucleotide, wherein each polynucleotide constituting the pair has anucleotide sequence shown in FIG. 1. Combination of a αCDR3polynucleotide and a βCDR3 polynucleotide differs according to eachclone. The nucleotide sequence of a pair of an αCDR3 bolynucleotide anda βCDR3 polvnucleotide in each clone are as shown in FIG. 1.

A polynucleotide having a nucleotide sequence complementary to a αCDR3polvnucleotide or a βCDR3 polynucleotide is also included in a αCDR3polynucleotide or a βCDR3 polynucleotide in addition, a degeneratesacuence of a αCDR3 polynucleotide a βCDR3 polynucleotide is alsoincluded in a αCDR3 polynucleotide or a βCDR3 poivnucleotide so long asit encodes the peptide shown in FIG. 1.

A polynucleotide having a nucleotide sequence identity of 70% or more,for example 75% or more, 80% or more, 85% or more, or 90% or more, forexample 92% or more, 94% or more, 96% or more, or 98% or more, to thatof a αCDR3 polynucleotide, is also included in a αCDR3 polynucleotide. Apolynucleotide having a nucleotide sequence identity of 70% or more, forexample 75% or more, 80% or more, 85% or more, or 90% or more, forexample 92% or more, 94% or more, 96% or more, or 98% or more, to thatof a βCDR3 polynucleotide, is also included in a βCDR3 olynucleotide.

A polynucleotide having a nucleotide sequence hybridizing toa nucleotidesequence of a αCDR3 polynucleotide under a stringent condition is alsoincluded in a αCDR3 polynucleotide. A polynucleotide having a nucleotidesequence hybridizing to a nucleotide sequence of a βCDR3 polynucleotideunder a stringent condition is also included in a βCDR3 polynucleotide,

Examples of stringent hybridization conditions include a condition wherehybridization is performed in a solution containing 5×SSC, 7% (w/v) SDS,100 μg/ml denatured salmon sperm DNA and 5×Denhardt' solution at 48-52°C., and then washin is performed in 0.1×SSC, 0.5×SSC, 1×SSC or 2×SSC ora condition where hybridization is performed in a solution containing250 MM NaCl, 25 mN trisodium citrate, 1% SDS, 50% formamide and 200μg/ml denatured salmon sperm DNA at 42° C., and then washing isperformed. in solution containing 15 inN NaCl, 1.5 mM trisodium citrateand 0.1% SDS.

In another aspect, the present invention provides peptide: encoded DvαCDR3 polynucleotides and βCDR3 polynucleotides (referred as “αCDR3peptide” and “βCDR3 peptide”, respectively). These peptides have theamino acid sequences shown in FIG. 1. Preferably, these peptides form apair of a αCDR3 peptide and a βCDR3 peptide corresponding to each cloneas shown in FIG. 1.

In the present specification, an amino acid sequence of a peptide isexpressed by conventional one-letter system or three-letter system.

A peptide encoded by variants of a αCDR3 polynucleotide or a βCDR3polynucleotide is also included in αCDR3 peptide or βCDR3 peptide. Apeptide having an amino acid sequence identity of 70% or more, forexample 75% or more, 80% or more, 85% or more, or 90% or more, forexample 92% or more, or 94% or more, to that of a αCDR3 peptide, is alsoincluded in a αCDR3 peptide. A peptide having an amino acid sequenceidentit_(y) of 70% or more, for example 75% or more, 80% or more, 85% ormore, or 90% or more, for example 92% or more, or 94% or more, to thatof a βCDR3 peptide, is also included in a βCDR3 peptide. In addition, apeptide having an amino acid sequence of a αCDR3 peptide in which one toseveral (for example, one, two, three, four or five) amino acids aresubstituted, deleted or added is also included in a αCDR3 peptide; and apeptide having an amino acid sequence of a βCDR3 peptide in which one toseveral (for example, one, two, three, four or five) amino acids aresubstituted, deleted or added is also included in a βCDR3 peptide. It isnoted that these variant peptides has similar properties to those of theoriginal αCDR3 peptides or βCDR3 peptides.

These polynucleotides and polypeptides can be prepared using chemicalmethods and/or biological methods well-known in the art.

In the present invention, the WT1 helper peptide is a peptide having anamino acid sequence shown in SEQ ID NO: 123 (Lys Arg Tyr Phe Lys Leu SerHis Leu Gln Met His Ser Arg Lys His) or a variant amino acid sequencethereof (These peptides are referred as “WT1₃₃₂ peptide”). WT1₃₃₂peptide may have a partial sequence or a variant sequence of WT1polypeptide. A peptide consisting of an amino acid sequence shown in SEQID NO: 123 or a variant sequence thereof is an example of such apeptide.

It is known that WT1₃₃₂ peptide has an ability to hind to aHLA-DPB1*15:01 molecule, HLA-DPB1*09:01 molecule, HLA-DPB1*05:01molecule, HLA-DRB1*04:05 molecule or a HLA-DRB1*15:02 molecule.

A variant sequence of the amino acid sequence shown in SEQ ID NO: 123 asabove mentioned refers to an amino acid sequence shown in SEQ ID NO: 123in which one to several (for example, one, two, three, four or five)amino acids are substituted, deleted or added. Or, a variant sequence ofthe amino acid sequence shown in SEQ ID NO: 123 as above mentionedrefers to an. amino acid sequence having an identity of 70% or more, forexample 75% or more, 80% or more, 85% or more, or 90% or more, to theamino acid sequence shown in SEQ ID NO: 123. Preferably, a peptidehaving the amino acid sequence shown in SEQ ID NO: 123 or a variantsequence thereof has a length of 25 amino acids or less. A peptidehaving a variant sequence of the amino acid sequence shown in SEQ ID NO:123 has similar properties to those of the peptide having the amino acidsequence shown in SEQ ID NO: 123.

In a further aspect, the present invention relates to a TCR genecontaining a αCDR3 polynucleotide and a βCDR3 polynucleotide belongingto any pair shown in FIG. 1. Such a TCR gene may be isolated from a CD4⁺T-cell specific to WT1₃₃₂ peptide, or may be prepared using well-knowngenetic engineering technology.

In a further aspect, the present invention relates to a CD4⁺ helperT-cell (referred as “TCR gene introduced CD4⁺ helper T-cell”) obtainedby introducing a TSR gene containing a αCDR3 polynucleotide and a βCDR3polynucleotide belonging to any pair shown in FIG. 1 into a CD4⁺ T-cell.A TCR, gene introduced CD4⁺ helper T-cell shows WT1₃₃₂-specific and HLAclass II-restricted proliferation and cytokine production.

A skilled person in the art can easily introduce a TCR gene containing aαCDR3 polynucleotide and a βCDR3 polynucleotide belonging to any one ofpairs shown in FIG. 1 into a CD4⁺ T-cell. For example, the introductionof a TCR gene can be done using various kinds of vectors,electroporation, or a gene gun, etc. A TCP gene to be introduced can bemodified for the purpose such as improvement of TSR expressionefficiency.

Therefore, in a further aspect, the resent invention provides a vectorcontaining a TCR gene containing a αCDR3 polynucleotide and a βCDR3oolvnucleotide belonging to any pair shown in FIG. 1.

Introduction of a TCR gene may be done by inserting a α-chain genecontaining a αCDR3 polynucleotide and β-chain gene containing a βCDR3polynucleotide into individual vectors, and introducing these vectorsinto a CD4⁺ T-cell.

Examples of CD4⁺ T-cells into which a TCR gene containing a αCDR3polynucleotide and a βCDR3 polynucleotide is introduced include CD4⁺T-cells derived. from a HLA-DRB1*15:01-positive subject, aHLA-DPB1*09:01-positive subject, a HLA-DPB1*05:01-positive subject, aHLA-DRB1*04:05-positive subject or- a HLA-DRB1*1502-positive subject,but not limited to them. In addition, CD4⁺ T-cells may be derived from asubject having a cancer, may be derived from a subject having no cancer(a healthy subject), or may be derived from a donor for bone marrowtransplantation.

In further aspect, the present invention also relates to aWT1₃₃₂-specific CD4⁺ helper T-cell comprising a TCR gene containing aαCDR3 polynucleotide and a βCDR3 polynucleotide belonging to any pairshown in FIG. 1.

Induction of a WT1-specific CTL can be enhanced using a TCRgene-introduced CD4⁺ helper T-cell. Particularly, induction ofWT1-specific CTL can be enhanced by co-culturing a TCR cene-introducedCD4⁺ helper T-cell and a peripheral mononuclear cell. Therefore, infurther aspect, the present invention provides a method for enhancingthe induction of a WT1-specific CTL, comprising co-culturing a TCRgene-introduced CD4⁺ helper T-cell and a peripheral mononuclear cell. Inanother aspect, the present invention relates to a WT1-specific CTLobtainable by said method.

Methods and conditions for co-culturing a TCR gene-introduced CD4⁺helper T-cell and a peripheral mononuclear cell are well known in theart. Such methods can be performed either in vivo or in vitro. One kindof a TCR gene-introduced CD4⁺ helper T-cell may be used for enhancingthe induction of a WT1-specific CTL. However, preferably two or morekinds of TCR gene-introduced CD4⁺ helper T-cells are used.

Examples of peripheral mononuclear cells used in the method forenhancing the induction of WT1-specific CTLs of the present inventioninclude peripheral mononuclear cells derived from aHLA-DRB1*15:01-positive subject, a HLA-DPB1*09:01-positive subject,HLA-DPB1*05:01-positive subject, a HLA-DRB1*04:05-positive subject or aHLA-DRB1*15:02-positive subject but not, limted to them. Preferably, theperipheral mononuclear cells and the CD4⁺ T-cells are those which havebeen obtained from a subject in which a cancer should be treated orprevented.

In the co-cultivation, it is preferable that WT1 ₃₃₂ peptide and/orother WT1 peptides co-exist. Examples of other WT1 peptides includethose which have ability to bind to a HLA-DRB1*15:01 molecule, aHLA-DPB1*09:01 molecule, a HLA-DPB1*05:01 molecule, a HLA-DRB104:05molecule or a HLA-DRB1*15:02 molecule, but not limited to them.

If necessary, the WT1-specific CTLs obtained by the method mentionedabove may be further cultured to make the cell numbers increase, andthen administrated to a subject, in order to treat or prevent a cancerin the subject. In such a treatment or prevention of a cancer, it ispreferable to co-administrate WT1₃₃₂ peptide and/or other WT1 peptides,By the action of the WT1-specific CTL, CTLs specific to other cancerantigens can also be induced.

A TCR gene-introduced CD4⁺ helper T-cell can damage cancer cellsexpressing WT1. Therefore, in further aspect, the present invention is amethod for the treatment or prevention of a cancer in a subject,comprising introducing a TCR gene-introduced CD4⁺ helper T-cell into thesubject.

In further aspect, the present invention provides a pharmaceuticalcomposition comprising a TCR gene-introduced CD4⁺ helper T-cell for thetreatment or prevention of a cancer, a use of a TCR gene-introduced CD4⁺helper T-cell for the manufacture of a medicament for the treatment orprevention of a cancer, and, a use of a TCR gene-introduced CD4⁺ helperT-cell for the treatment or prevention of a cancer.

As used herein, “treatment” of a cancer refers not only to the treatmentof a cancer such as the inhibition of progress of a cancer, thereduction of a cancer and the destruction of a cancer, but also to theprevention of recurrence of a cancer.

Examples of subjects in which a cancer is treated or prevented include aHLA-DRB1*15:01-positive subject, a HLA-DFB1*0901-positive subject, aHLA-DPB1*0501-positive subject, a HLA-DRB1*04:05-positive subject or aHLA-DRB1*15:02-positive subject, but not limited to them, above subjectis not limit ed to a cancer patient, and may be a person not having acancer (including a healthy person), or may be a donor for bone marrowtransplantation.

Embodiments of the method for the treatment or prevention, thepharmaceutical composition, and the use as mentioned above are describedbelow. However, the embodiments are not limited to those, First, CD4⁺T-cells are taken from a peripheral blood of a cancer patient who needsa treatment, and a TCR gene containing a αCDR3 polynucleotide and aβCDR3 oolynucleotide is introduced into the CD4⁺ T-cells to obtain TCRgene-introduced CD4⁺ helper T-cells. The TOE mete introduced CD4⁺ helperT-cells thus obtained are administered to the cancer patient. Before theadministration, the TCR gene-introduced CD4⁺ helper T-cells can becultured and proliferated under appropriate conditions to obtain asufficient number of cells, and then they can be administered to thecancer patient.

Either one kind of TCP gene-introduced CD4⁺ helper T-cell or two or morekind of TCP gene-introduced CD4⁺ helper T-cells may be administered.From the viewpoint of improvement of treatment or prevention effect, itis preferable that two or more kinds of TCR gene-introduced CD4⁺ helperT-cells are administered to a subject.

In case that TCP oene-introduced CD4⁺ helper T-cells are administered toa subject, a physician can appropriately decide conditions such as thenumber of cells to be administered, the frequency of the administration,the interval of the administration. For example, TCR gene-introducedCD4⁺ helper T-cells may be administered only once, or administeredseparately several times. Typically, in case of an adult subject, thenumber of the TCR gene-introduced CD4⁺ helper T-cells per dose is in arange between about 10⁹ and about 10¹¹, but not limited these numbers.

In the treatment or prevention method, the pharmaceutical compositionand the use described above, it is preferable that WT1₃₃₂ peptide and/orother WT1 peptides are co-administered. A physician cart appropriatelydecde amount and frequency of administration of WT1₃₃₂ peptide and/orother WT1 peptides. In addition, other anti-cancer therapies orpreventions may be combined.

The method for the treatment or prevention, the pharmaceuticalcomposition, and the use described above can be applied to various kindsof cancers, but not limited to, for example, hematologic malignancies,such as acute myelocytic leukemia, acute lymphocytic leukemia, malignantlymphoma, multiple myeloma, chronic myelocytic leukemia, myelodysplasticsyndrome, and recurrence after the hamatopoietic stem celltrnasplantation of the same type; solid cancers, such as tongue cancer,gingival cancer, mouth floor cancer, pharyngeal cancer, larynx cancer,salivary gland cancer, and thyroid cancer; thoracic cancers, such asbreast cancer, lung cancer, and thymic cancer; gastrointestinal cancers,such as colon cancer, small intestine cancer, gastric cancer, pancreaticcancer, liver cancer, bile duct cancer, gastrointestinal endocrinetumor, and gastrointestinal carcinoid; cancers of urinary and genitaltract, such as renal cancer, urothelial cancer, germinoma, Wilms' tumor,prostate cancer, uterine body cancer, cervical cancer, uterine sarcoma,and ovarian malignancy; musculoskeletal malignancies, such as primarymalignancy of bone (e.g., osteosarcoma and Ewing's sarcoma) and softtissue sarcoma; and other cancers, such as skin cancer, neuroblastoma,malignant glioma (glioblastoma), primary malignant lymphoma of thecentral nervous system, medulloblastoma, and PNET.

The CDR3 regions are the most diverse portions and are the mostresponsible parts for the specificity of antigen recognition. Thus, thesequences of the αCDR3 polynucleotides, the βCDR3 polynucleotides, theαCDR3 peptides, and the βCDR3 peptides of the present invention areconsidered peculiar to the CD4⁺ helper Tcells specific to WT1₃₃₂peptide. Therefore, in case that a polynucleotide encoding a CDR regionof a α-chain and a β-chain, or a peptide corresponding to the CDR regionhave the sequence of the polynucleotide or the peptide of the presentinvention, the CD4⁺ helper T-cell is considered to be specific to WT1₃₃₂peptide.

For example, (i) a DNA chip comprising one or more kinds of αCDR3polynucleotides, (ii) a DNA chip comprising one or more kinds of βCDR3polynucleotides, or (iii) a DNA chip comprising both one or more kindsof αCDR3 polynucleotides and one or more kinds of βCDR3 polynucleotidescan be used to measure the frequency of CD4⁺ helper T-cells specific toWT1₃₃₂ peptide in a sample. Particularly, a sample is prepared by lysingcells in a specimen obtained from a subject and extracting nucleicacids, and the sample is contacted with the DNA chip.

For example, in case that a sample is contacted with the chip (andhybridization is found at any position, the same sample is contactedwith the chip (ii) to confirm whether hybridization is found or not.Then, in case that any hybridization in the chip (i) and anyhybridization in the chip (ii) occur with any αCDR3 polynucleotide andany βCDR3 polynucleotide which constitute any pair shown in FIG. 1, itcan be judged that a CD4⁺ helper T-cell specific to WT1₃₃₂ peptidehaving a functional TCR exists in the sample. Using the chip (III), theabove process can be done in one step.

A DNA chip may be in any form such as a microchip and a microarray.These chips can be prepared by a well-known method. For example, αCDR3polynucleotides and βCDR3 polynucleotides can be immobilized on a glasssubstrate by a well-known method. It is preferable that a label whichcan indicate presence or absence of hybridization and amount of thehybridization is attached to DNAs in a sample or DNA sequences on achip.

Not only a DNA chip but also techniques such as southern blotting,northern blotting, colony hybridization can be used to measure frequencyof CD4⁺ helper T-cells specific to WT1₃₃₂ peptide in a sample.

In addition, a αCDR3 peptide and a βCDR3 peptide can be used to obtainan antibody to a CD4⁺ helper T-cell specific to WT1₃₃₂ peptide. A CD4⁺helper T-cell specific to WT1₃₃₂ peptide can be detected using such anantibody. A receptor of a CD4⁺ helper T-cell specific to WT1₃₃₂ peptidecan also be stimulated using such an antibody. Such stimulation can bedone either in vivo or in vitro.

A chip comprising aODR3 peptides, a chip comprising βCDR3 peptides, or achip comprising both αCDR3 peptides and βCDR3 peptides can also be usedto detect an antibody to a CD4⁺ helper T-cell specific to WT1₃₃₂peptide.

A chip comprising these peptides can be prepared using a well-knownmethod. It is preferable to add a label which can determine presence orabsence of a specific binding to peptides in a sample or peptides on achip.

A chip comprising antibodies to αCDR3 peptides and/or βCDR3 peptides canalso be used to determine kind and amount of βCDR3 peptides and/or βCDR3peptides in a sample, or to determine kind and amount of CD4⁺ helperT-cells specific to WT1₃₃₂ peptide in a sample.

A chip to which these antibodies are immobilized can be prepared using awell-known method. It is preferable to add a label which can determinepresence or absence of a specific binding to peptides in a sample orantibodies on a chip.

Description of Sequences

SEQ ID NOs: 1 to 59 are nucleotide secuences encoding CDR3 contained inTCR of CD4⁺ helper T-cell clones.

SEQ ID NOs: 60 to 118 are amino acid sequences of CDR3 contained in TCRof CD4⁺ helper T-cell clones.

SEQ ID NO: 119 is a reverse primer for amplifying TCRα chain.

SEQ TD NO: 120 is a reverse primer for amplifying TCRβ chain.

SEQ ID NO: 121 is a reverse primer for amplifying TCRβ chain.

SEQ ID No: 122 is a primer for determining CDR3 nucleotide sequences.

SEQ ID NO: 123 is an amino acid seauence of WT1₃₃₂ peptide,

SEQ ID NO: 124 is an amino acid sequence of HIV peptide.

SEQ ID NO: 125 is an amino acid sequence of a variant of a naturallyoccurring WT1 peptide.

The present invention is described more particularly and more concretelyby showing examples below. However, it should not be construed thatexamples limit the scope of the present invention.

EXAMPLE 1

Example 1 Establishment of WT1₃₃₂-specific CD4⁺ T-cell clones andisolation and sequencing of T-cell receptor (TCR) genes

The experimental procedures were as follows.

(1) Method of establishing WT1₃₃₂-specific CD4⁺ T-cell clones

(i) Peripheral blood mononuclear cells (PBMCs) derived from a healthysubject are harvested and seeded into 24-well plates at 3×106cells/well. X-VIVO 15 medium supplemented with 10% AB serum and 40 IU/mlIL-2 is used as a medium.

(ii) WT1₃₃₂ peptide is added to the above i at a final concentration of20 μg/ml and the cells are cultured for 7 days,

(iii) After 7 days, the cells are collected and prepared with X-VIVO 15medium supplemented with 10% AB serum so that the cell density is 1×107cells/mi, and then, seeded by 100 μL each into 96 well, round bottomplates.

(iv) WT1₃₃₂ peptide, BD GolgiStop™ (BD Bioscience) and CD28/CD49dCostimulatory Reagent (BD Bioscience) are added to X-VIVO 15 mediumsupplemented with 10% AB serum at final concentrations of 40 μg/ml, 4μg/ml, and 4 μg/ml, respectively.

(v) The above iv is added by 100 μL each to the above iii.

(vi) Anti-human C154-APC-labeled antibody (BD Bioscience) is added by 10μl each to the above v and the plates are incubated in 5% CO₂ incubatorfor 6 hours at 37° C.

(vii) After incubation, the cells are collected and stained withanti-human CD4-APC-H7-labeled antibody (BD Bioscience) and anti-humanCD3-Pacific Blue-labeled antibody (BD Bioscience) as well as 7-AAD(eBioscience) for removing dead cells.

(viii) PSMCs are harvested from 3 healthy sublects, mixed, irradiatedwith 30 Gy of γ-ray, and prepared with X-VIVO 15 medium supplementedwith 10% AB serum at a final concentration of 10%, IL-2 at a finalconcentration of 100 IU/ml, and PHA at a final concentration of 3 μg/mlso that the cell density is 1×106 cells/ml. These prepared cells areseeded by 100 μL each into 96 well, round bottom plates, (ix)7-ADD-CD3⁺CD4⁺CD154⁺ cell fraction, i.e., a fraction containingWT1₃₃₂-specific CD4⁺ T-cells is single-cell sorted into each well of theabove viii using FACSAria cell sorter.

(x) After culture for 10-14 days, the proliferated cells in each wellare used as independent CD4⁺ T-cell clones.

(2) Screening of WT1₃₃₂-specific CD4⁺ T-cellclones

(i) Each CD4⁺ T-cell clone of the above (1)-x is prepared with X-VIVO 15medium supplemented with 1% AB serum so that the cell density is 3×105cells/ml.

(ii) Autologous PBMCs pulsed with WT1₃₃₂ or not pulsed with any peptidesare irradiated with 30 Gy of γ-ray and prepared with X-VIVC 15 mediumsupplemented with 1% AB serum so that the cell density is 1×106cells/ml.

(iii) The above (2)-i and ii are seeded by 100 μL each into 96 well,round bottom plates.

(iv) After culture for 2 days, 3H-thymidine is added to each well at 1μCi/well.

(v) After 18 hours, the 3E-thymidine incorporated into each CD4⁺ T-cellclone is measured and the CD4⁺ T-cell clones showing WT1₃₃₂

specific proliferative response are selected. These selected clones areused as WT1₃₃₂-specific CD4⁺ T-cell clones.

(vi) The culture of the WT1₃₃₂ specific CD4⁺ T-cell clones is performedwith stimulation of the WT1₃₃₂ specific CD4⁺ T-cell clones byco-culturing with PMCs that were prepared by irradiating autologousPBMCs pulsed with. WT1₃₃₂ at a frequency of once per 1-2 weeks or sowith 30 Gy of γ-ray.

(3) Isolation of TCR genes using, 5′-RACE (Rapid Amplification of cDNAEnd) method

(i) WT1₃₃₂ specific CD4⁺ T-cell clones are cultured for 10 days or morefrom the last stimulation. This is to prevent contamination with T-cellscontained in autologous PBMCs that are used for the stimulation.

(ii) The WT1₃₃₂-specific CD4⁺ T-cell clones are pelleted, TRIzol reagent(Invitrogen) is added thereto, and RNA is extracted according to itsmanual.

(iii) cDNAs are synthesized from the RNA extracted in the above (3)-iiusing SMARTer™ RACE cDNA Amplification Kit Clontech).

(iv) TCR α-chain and β-chain genes are amplified by using the cDNAssynthesized in the above (3)-iii as templates In regard to used primers,UPM primer included in MARTer™ RACE cDNA Amplification Kit was used as aforward primer and the following TCR-specific primers were used asreverse primers:

Cα3′UTR-primer: (SEQ ID No: 119)5′-CAC AGG CTG TCT TAC AAT CTT GCA GAT C-3′ Cβ1-3′UTR-primer:(SEQ ID No: 120) 5′-CTC CAC TTC CAG GGC TGC CTT CA-3′ Cβ2-3′UTR-primer:(SEQ ID No: 121) 5′-TGA CCT GGG ATG GTT TTG GAG CTA-3′.

(v) The amplification of the TCR genes was performed using KOD FXavailable from ToYoBo under conditions of 94° C., 3 min->(98° C., 10sec->68° C., 1 min)×35 cycles.

(vi) The size of PCR products are confirmed using agarose gelelectrophoresis and bands of near 1 kbp are cut from gel and purified.

(vii) After adenines are added to the PCR products purified in the above(3)-vi using Tag polymerase, the resultants are ligated into pCR 2.1vectors.

(viii) HST02 competent cells are transformed with the above plasmids arepurified from single colonies, and then, sequenced.

(ix) The sequence analysis is performed using the InternationalImmunogenetics Information Sys Nem(http://www.imgt.org/IMGT_vquest/vquest?livret=0&Option=humanTcR) andeach TCR gene is identified.

With regard to the above (3) “isolation of TCR renes using 5′-RACE(Rapid Amplification of cDNA End) method”, the detailed experimentalprocedure is shown below.

(3-1) RNA extraction

RNA extraction from T-cell clones was performed using TRIzol Reagent(Invitrogen). As for T-cell clones used, the clones cultured withoutantigen-stimulation art the presence of IL-2 over 3 weeks were preparedfor the purpose of preventing contamination with feeder cells.

(3-2) Cloning of full-length TCR (T-cell receptor) cDNA using 5′-RACE(Rapid Amplification of cDNA Ends) method

For cloning of TCR α/β, SMARTerT™ RACE cDNA Amplification Kit (Ciontech)was used. Firstly, 5′-RACE reaction was performed according to itsmanual, and thereby 1st strand cDNA was synthesized. Then, in order toobtain ti full-length TCR α-chain and β-chain cDNAs, PCR reaction wasperformed by using reverse primers specific to each of 3′UTRs(Untlansrated Regions) and, unlversal primer (UPM) which is included inthe kit and using the synthesized 1st strand cDNA as a template. Theused primers are as follows.

Cα 3′UTR-RACE-primer: (SEQ ID No: 119) CACAGGCTGTCTTACAATCTTGCAGATCCβ1 3′UTR-RACE-primer: (SEQ ID No: 120) CTCCACTTCCAGGGCTGCCTTCACβ2 3′UTR-RACE-primer: (SEQ ID No: 121) TGACCTGGGATGGTTTTGGAGCTA

Further, FOR reaction was performed in the following reaction solutioncomposition using KOD FX (TOYOBO).

TABLE 1 2x PCR buffer for KOD FX 12.5 μl 2 mM dNTPs 5.0 μl 10x UPM 2.5μl 10 μM reverse primer 1.0 μl Template DNA 1.0 μl KOD FX (1.0 U/μl) 0.5μl distilled water up to 25 μl Volume of the reaction solution 25 μl PCRcycle is as follows: 94° C., 2 min −> (98° C., 10 sec −> 68° C., 1 min)× 35 cycles −> 15° C., hold

After the PCR reaction, 1.0% agarose gel electrophoresis was performed,single bands of near 900-1000 bp were cut, and the PCR products werepurified with 50 μl of distilled water using QTAquick Gel Extraction Kit(QIAGEN). It is necessary to add adenine to both ends of the PCRproducts for TA-cloning. The addition of adenine was performed usingPlatinum Tag DNA polymerase (invitrocen) as follows.

(1) 2× reaction solution shown in the table below is prepared.

TABLE 2 10x PCR buffer 10 μl 2 mM dNTPs 10 μl 25 mM MgCl2 8.0 μlPlatinum Taq polymerase 1.0 μl 21 μl of distilled water is added so thata total volume is 50 μl.

(2) 2× reaction solution is incubated at 95° C. for 5 minutes.

(3) The purified PCR products are added thereto.

(4) The resultants are incubated at 72° C. for.10 minutes.

PCR products added with adenines were purified and concentrated byethanol precipitation, and then, inserted into pCR 2.1 vectors(invitrogen) using DNA Ligation Kit, Mighty Mix>(TaKaPa). pCR 2.1vectors comprising the POP products were introduced into HST02 competentcells by transformation and cloned.

(3-3) Purification of plasmids comprising full-length TCR α-chain andβ-chain cDNAs

The transformed HST02 competent cells were plated on ampicillin/LBplates and incubated at 37° C. Then, single colonies were picked intoampicillin/LB liquid medium, and incubated 37° C. while being stirred at200 rpm. Then, plasmids were purified from the Escherichia coli solutionusing AUTOMATIC DNA ISOLATION SYSTEM PI-50 (KURABO).

(3-4) Determination of CDR3 sequence of TCR by seguencing

For sequencing of the purified plasmids, BigDye® Terminator v3.1 CycleSequencing Kit (Applied Biosystems) was used. In addition, M13 reverseprimer:

(SEQ ID No: 122) caggaaacagctatgac

was used. For analysis of TCR and CD3, IMGT/V-QUEST(http://www.imgt.org/IMGT_vquest/share/textes/) was utilized.

The determined nucleotide and amino acid sequences of CDR3 are shown inFIG. 1. In some of the clones, there were 2 kinds of α-chain and 2 kindsof CDR 3 sequences.

EXAMPLE 2

Example 2 introduction of T-cell receptor (TCR) genes derived from aWT1₃₃₂-specific CD4⁺ T-cell to human CD4⁺ T-cells

It was confirmed that human CD4⁺ T-cell transduced with T-cell receptor(TCR) genes derived from a WT1₃₃₂-specific CD4⁺ T-cell showedproliferative response and production of cytokines in a WT1₃₃₂-specificand HLA class II-restricted manner.

TCR genes shown in Table 3 were isolated from clone 9 which is the CD4⁺T-cell clone which specifically recognizes WT1₃₃₂ in anHLA-DPB1*05:0l-arestricted manner, These TCR genes were transduced intoCD4⁺ T-cells derived from peripheral blood of healthy subjects by usinglentivirus vectors, and the response to WT13₃₃₂ was examined hy usingthe productions of cytokines (interferon-γ and IL-2) as indicators(FIGS. 2A and B), In addition, CD4⁺ T-cells transduced with lentivirusvectors not carrying TCR genes (indicated as mock) were used as acontrol, CD4⁺ T-cells transduced with WT1₃₃₂-specific TCR genes(referred as “WT1₃₃₂-TCR-transduced CD4⁺ T-cells” in the section orExamples) produced INF-γ and IL-2 in response only to W1₃₃₂, i.e., in aWT1₃₃₂-specific manner. On the other hand, the mock-transduced CD4⁺T-cells did not show WT1₃₃₂-specific production of cytokines.

The effect of the concentration of WT1₃₃₂ peptide on the expression ofcytokine by WT1₃₃₂-TCR-transduced CD4⁺ T-cells was examined,WT1₃₃₂-TCR-transduced CD4⁺ T-cells were stimulated with variousconcentrations of W1₃₃₂ peptide for 4 hours, and intracellular cytokinestaining assay was performed to examine the ratio of TNF-α-producingCD4⁺ T-cells to CD4⁺ T-cells. The results are shown in FIG. 2C. Theproduction of the cytokine was WT1₃₃₂ peptide concentration-dependentand ED50 was 4.85 μM.

When a proliferation potency of the WT1₃₃₂-TCR-transduced CD4⁺ T-cellswas examined, a WT1₃₃₂-specific, strong proliferation potency was foundand the proliferative response was markedly inhibited by anti-HLA-DPantibody (FIG. 2D).

Next, the proliferative response and IFN-γ production of the WT1₃₃₂TCR-transduced CD4⁺ T-cells to autologous PBMCs pulsed with WT1₃₃₂peptide, autologous PENCs pulsed with full-length WT1 protein,autologous PBMCs pulsed with truncated. WT1 protein (not comprisingW1₃₃₂ sequence), PEMCs pulsed with the lysate of PHA-blast, PBMCs pulsedwith the lysate of leukemia cell line TF-1 expressing WT1, and PEMOspulsed with the lysate of leukemia cell line K562 expressing WT1 wereexamined, The cell proliferation was measured by [3H]-thymidineincorporation, and IFN-γ was measured by ELISA. The results are shown inFIG. 2E and 2F, respectively. It was found that the proliferation andIFN-γ production of the WT1₃₃₂ TCR-transduced CD4⁺ T-cells were markedlystimulated by PBMCs pulsed with the lysate of leukemia cell lines (TF-1and K562) expressing WT1 and also stimulated by autologous PBMCs pulsedwith WT 1₃₃₂ peptide and autologous PEMCs pulsed with full-length WT1protein.

Further, the production of various cytokines that responded to WT1₃₃₂peptides of WT1₃₃₂-TCR-transduced CD4⁺ T-cell lines prepared similarlyto those of Example 2, that were derived from 3 healthy (HLA-DPB1*0501positive) donors (i.e., three kinds of cell lines), was also examined.The mean values of the cytokine-producing abilities of the three kindsof cell lines are shown in FIG. 2G. Th1-type cytokines such as IL-2,IFN-γ, TNF-α and GM-CSF were produced in large amount.

TABLE 3 TCR genes isolated form clone 9 V gene J segment D geneCDR3 sequence Va TRAV13- TRAJ53* — CAENSGGSNYKLTF 8.2 2*01 01(SEQ ID No: 73) Vb TRAB6- TRBJ1- TRBD01* CASTAGASDQPQHF  13.3 1*01 5*0101 (SEQ ID No: 74)

EXAMPLE 3

Example 3 Enhanced induction of WT1-specific CTLs by human CD4⁺ T-cellstransduced with. TCR genes derived from WT1₃₃₂-specific CD4⁺ T-cells

Generally, it is known that CD4⁺ T-cell serves as helper T-cell and isimportant for introduction and maintenance of CD8⁺ T-cells (CTLs) thatare the primary effector cells that attack cancer cells. Thus, it wasexamined whether WT1₃₃₂-TCR-transduced CD4⁺ T-cells enhanced theinduction of WT1-specific CTLs.

PBMCs of HLA-A*24:02 and HLA-DPB1*05:01-positive healthy subjects weremixed with WT1₃₃₂-TCR-transduced CD4⁺ T-cells prepared from the samehealthy subjects at the ratio of 10:1 and 5:1 (indicated as 1:0.1 and1:0.2 in FIG. 3) and incubated for 1 week in the presence of a modifiedWT1₂₃₅ peptide (wherein M, the second amino acid of natural WT1 peptidebinding to HLA-A*24:02 molecule, was modified into Y (CYTWNQMNL)(SEQ IDNo:125)) that is an HLA-A*24:02-restricted CTL epitope derived from WT1and WT1₃₃₂. Then, the resultant was stimulated again with the modifiedWT1₂₃₅ peptide (wherein the binding ability to HLA-A*24:02 molecule wereenhanced) and further incubated for 1 week. No IL-2 was added in aseries of cultures in order to correctly evaluate the help activity ofCD4⁺ T-cells, After 2 weeks cultures in total, it was examined whetherthe induction of WT1-specific CTLs was enhanced by the WT1₃₃₂TCR-transduced CD4⁺ T-cells by using frequencies of CD8⁺ T-cells,modified WT1/HLA-A*24:02 tetramer-positive CD4⁺ T-cells, and modifiedWT1₂₃₅-specific interferon-y (INF-γ)-expressing CD8⁺ T-cells asindicators. As a result, the frequency of CD8⁺ T-cells was significantlyhigher when co-cultured with the WT1₃₃₂-TCR-transduced CD4⁺ T-cells ascompared when cultured with the mock-transduced CD4⁺ T-cells as acontrol (FIG. 3A). In addition, in regard to the modifiedWT1₂₃₅/HLA-A*24:02 tetramer positive CD8⁺ T-cells that are el-specificCTLs, a clearly positive population was found when co-cultured with theWT1₃₃₂-TCR-transduced CD4⁺ T-cells, however, it was not found in thecontrol (FIG. 3B). Calculating the cell number of the WT1-specific CTLspresent in 100,000 lymphocytes from these results, the cell number wasabout 28 times higher when co-cultured with the WT1₃₃₂-TCR-transducedCD4⁺ T-cells compared with the control (FIG. 3C). Likewise, thefrequency of CD8⁺ T-cells expressing INF-γ by the stimulation with themodified WT1₂₃₅ was also significantly high when co-cultured with theWT1₃₃₂-TCR-transduced CD4⁺ T-cells (FIG. 3D). From the above, it wasrevealed that the WT1₃₃₂-TCR-transduced CD4⁺ T-cells enhanced theinduction of WT1-specific CTLs.

EXAMPLE 4

Example 4 HLA-DPB1*05:01-restricted damage of WT1-expressing leukemiacells by human CD4⁺ T-cells transduced with TCR genes derived fromWT1₃₃₂-specific CD4⁺ T-cells

Next, the cytotoxic activity, i.e., killing activify, ofWT1₃₃₂-TCR-transduced CD4⁺ T-cells was evaluated

Firstly, HLA-DPB1*05:01 gene was isolated and transfected into leukemiacell line TF-1 expressing WT1 to prepare HLA-DPB1*05:01-positive TF-1cells. As shown in FIG. 4A, the WT1₃₃₂-TCR-transduced CD4⁺ T-cellsstrongly damaged HLA-DPB1*05:01-positive TF-1 cells, however, they didnot exhibit cytotoxic activity on HLA-DPB1*05:01-negative TF-1 cells.Then, in order to confirm whether this cytotoxic activity isWT1-specific, B-LCL(+) was prepared by overexpressing WT1 gene inHLA-DPB1*05:01-positive B-LCL cells not expressing WT1 (indicated asB-LCL(−)), and these cells were used as target cells to evaluate thecytotoxic activity of WT1₃₃₂-TCR-transduced CD4⁺ T-cells. As shown inFIG. 4B, B-LCL(+) was strongly damaged by the WT1₃₃₂-TCR-transduced CD4⁺T-cells, however, B-LCL(−) was not damaged. From these results, it wasrevealed that the WT1₃₃₂-TCR-transduced CD4⁺ T-cells had theHLA-DPB1*05:01-restricted and WT1-specific cytotoxic activity. Further,the cytotoxic activity of the WT1₃₃₂-TCR-transduced CD4⁺ T-cells wasconfirmed by using leukemia cell line C2F8 which wasHLA-DPB1*05:01-postive and expressed WT1 (FIG. 4C).

Next, it was examined whether WT1₃₃₂-TCR-transduced CD4⁺ T-cells exertedthe cytotoxic activity via the granzyme B and perforin pathway. Highexpressions of granzyme B and perforin were found in theWT1₃₃₂-TCR-transduced CD4⁺ T-cells (FIG. 4D).

WT1₃₃₂-TCR-transduced CD4⁺ T-cells and CD4⁺ T-cells similarly treatedwith mock-vector (mock-transduced CD4⁺ T-cells) were cultured withHLA-DPB1*05:01-positive TF-1 cells pulsed with WT1₃₃₂ peptide orHLA-DPE1*05:01-positive TF-1 cells not pulsed with WT1₃₃₂ peptide for 5hours in the presence of anti-CD107a-APC-monoclonal antibody. Then,IFN-γ-staining was performed and the resultants were subjected to Flowcytometry. The co-expression of IFN-γ and CD107a was found in theWT1₃₃₂-TCR-transduced CD4⁺ T-cells only when the WT1₃₃₂-TCR-transducedCD4⁺ T-cells were incubated with HLA-DPB1*05:01-positive TF-1 cellspulsed with WT1₃₃₂ peptide (FIG. 4E). This shows that the degranulationoccurs in the WT1₃₃₂-TCR-transduced CD4⁺ T-cells.

To confirm whether the cytotoxic activity of the WT1₃₃₂-TCR-transducedCD4⁺ T-cells was dependent on the granzyme B/perforin pathway,HLA-DPB1*05:01-positive TF-1 cells pretreated with 100 μM of agranzyme-inhibitor, Ac-IETD-Cho, were used as target cells. TheHLA-DPB1*05:01-positive TF-1 cells were pretreated with 100 μM ofAc-IETD-Cho or DMO (control) for 2 hours, then labeled with ⁵¹Cr, andincubated with WT1₃₃₂-TCR-transduced CD4⁺ T-cells, and ⁵¹Cr releasingassay was performed. The cytotoxic activity of the WT1₃₃₂-TCR-transducedCD4⁺ T-cells on HLA-DRB1*05:01-positive TF-1 cells pretreated withAc-IETD-Cho was markedly lower compared with the cytotoxic activity onTF-1 cells pretreated with DMSO (FIG. 4F).

Considering these results together, it was confirmed that theWT1₃₃₂-TCR-transduced CD4⁺ T-cells obtained by the present inventiondirectly recognized HLA-DPB1*05:01-positive leukemia cells expressingWT1 and damaged them via granzyme B/perforin pathway.

EXAMPLE 5

Example 5 Anti-tumor effect in NOG® Mouse by human CD4⁺ T-cellstransduced with TCR genes derived from WT1₃₃₂-specific CD4⁺ T-cells

WT1-expressing HLA-DPB1*05:01-positive human leukemia cells C2F8 (5×104cells) were transferred to NOG® mice (7 mice) via tail vein. Next day,as an experimental group, human CD4⁺ T-cells transduced withHLA-DPB105:01-restricted WT1₃₃₂-specific TCR genes (SEQ ID Nos: 14 and15) (5×106 cells) and T-cell-depleted human peripheral blood mononuclearcells (2×106 cells) from the same subject as antigen-presenting cellswere transferred to the above NOG® mice (3 mice). As a control, humanCD4⁺ T-cells transduced with the control vector (5×106 cells) andT-cell-depleted human peripheral blond mononuclear cells (2×106 cells)from the same subject as antigen-presenting cells were transferred tothe above NOG® mice (4 mice).

After 1 and 2 weeks, human CD4⁺ T-cells transduced withHLA-DPB1*05:01-restricted WT1₃₃₂-specific TCR genes (5×106 cells) weretransferred to the mice of the experimental group via tail vein. HumanCD4⁺ T-cells transduced with control vector (5×106 cells) weretransferred to the control mice via tail vein. Then, the survival ofmice was examined.

The results are shown in FIG. 5. Since the survival rate of the mice ofthe experimental aroup exceeded the survival rate of the mice ofcontrol, it was shown that HLA-DPB1*05:01-restricted WT1₃₃₂-specificTCR-tranduced human CD4⁺ T-cells had an anti-tumor effect in vivo.

INDUSTRIAL APPLICABILITY

The present invention can be used in the fields of pharmaceuticals fortreating or preventing cancer, of reagents for cancer research, and ofcancer test kits or reagents, and the like.

1. A polynucleotide (referred as “αCDR3 polynucleotide”) having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 8, 10, 11, 13, 14, 16, 18, 20, 22, 23, 25, 27, 28, 30, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 56 and 58, wherein said polynucleotide encodes CDR3 of α-chain of TCR of a CD4⁺ helper T-cell specific to a WT1 helper peptide (WT1₃₃₂ peptide) having an amino acid sequence shown in SEQ ID NO: 123 or a variant sequence thereof.
 2. A polynucleotide (referred as “βCDR3 polynucleotide”) having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 7, 9, 12, 15, 17, 19, 21, 24, 26, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57 and 59, wherein said polynucleotide encodes CDR3 of β-chain of TCR of a CD4⁺ helper T-cell specific to WT1₃₃₂ peptide,
 3. A pair of a αCDR3 polynucleotide and a βCDR3 polynucleotide, wherein each polynucleotide has the following nucleotide sequence: α CDR3 polynucleotide βCDR3 polynucleotide SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 3 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 12 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 15 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 24 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 4 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 57 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59

provided that said sequence may be a complementary sequence or a degenerate sequence thereof.
 4. A TCR gene comprising the αCDR3 polynucleotide and the βCDR3 polynucleotide in any one of pairs according to claim
 3. 5. The TCR gene according to claim 4 obtainable from a CD4⁺ T-cell specific to WT1₃₃₂ peptide.
 6. A method for producing a CD4⁺ helper cell specific to WT1₃₃₂ peptide, comprising introducing the TCR gene according to claim 4 or 5 into a CD4⁺ T-cell.
 7. A CD4⁺ helper T-cell obtainable by the method according to claim
 6. 8. A vector comprising a TCR gene which comprises the αCDR3 polynucleotide and the βCDR3 polynucleotide in any one of pairs according to claim
 3. 9. The method according to claim 6 wherein said introduction is performed using a vector comprising a TCR gene which comprises the αCDR3 polynucleotide and the βCDR3 polynucleotide in any one of pairs of a αCDR3 polynucleotide and a βCDR3 polynucleotide, wherein each polynucleotide has the following nucleotide sequence: α CDR3 polynucleotide βCDR3 polynucleotide SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 3 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 12 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 15 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 24 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 4 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 32 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 57 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59

provided that said sequence may be a complementary sequence or a degenerate sequence thereof.
 10. A method for enhancing the induction of a WT1 specific CTL, comprising co-culturing the CD4⁺ helper T-cell according to claim 7 and a peripheral mononuclear cell.
 11. A WT1-specific CTL obtainable by the method according to claim
 10. 12. (canceled)
 13. A pharmaceutical composition for the treatment or prevention of a cancer, comprising the CD4⁺ helper T-cell according to claim
 7. 14. (canceled)
 15. A DNA chip comprising a αCDR3 polynucleotide free ed as “αCDR3 polynucleotide”) having, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 8, 10, 11, 13, 14, 16, 18, 20, 22, 23, 25, 27, 28, 30, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 56 and 58, wherein said polynucleotide encodes CDR3 of α-chain of TCR of a CD4⁺ helper T-cell specific to a WT1 helper peptide (WT1₃₃₂ peptide) having an amino acid sequence shown in SEQ ID NO: 123 or a variant sequence thereof, or a βCDR3 polynucleotide (referred as “βCDR3 polynucleotide”) having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 7, 9, 12, 15, 17, 19, 21, 24, 26, 29, 32, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 57 and 59, wherein said polynucleotide enclodes CDR3 of β-chain of TCR of a CD4⁺ helper T-cell specific to WT1₃₃₂ peptide, or both of the αCDR3 polynucleotide and the βCDR3 polynucleotide.
 16. A method for measuring the frequency of CD4⁺ helper T-cell specific to WT1₃₃₂ peptide in a sample, comprising using the DNA chip according to claim
 15. 17. An αCDR3 peptide encoded by any one of the αCDR3 polynucleotides according to claim
 1. 18. A βCDR3 peptide encoded by any one of the βCDR3 polynucleotides according to claim
 2. 19. A pair of peptides encoded by any one of pairs of the polynucleotides according to claim
 3. 20. A chip comprising the peptide according to claim 17 or 18, or the pair of the peptides according to claim
 19. 21. An antibody against any one of the peptides according to any one of claims 17 to
 19. 22. A method for measuring the frequency of CD4⁺ helper T-cell specific to WT1₃₃₂ peptide in a sample, comprising using the antibody according to claim
 21. 